1. Field of the Invention
Immunoassays have shown themselves to be extremely versatile in allowing for methods to determine the presence of a particular substance, even when a wide variety of other materials of similar or different structure are present in the unknown sample. The immunoassays rely on the ability of an antibody to specifically detect or bind to an haptenic organic compound, while not interacting with other compounds. The divalent nature of the antibody and its high molecular weight, 150,000 or greater, allow for a sufficient change in the compound or environment of the compound to permit a discrimination between a compound which is bound and a compound which is not bound to antibody. Among various immunoassays involving antibodies are radioimmunoassay, spin immunoassays, reagents for which are available under the trademark FRAT, supplied by Syva Company; homogeneous enzyme immunoassay, reagents for which are available under the trademark EMIT, supplied by Syva Company; and hemeagglutination.
The enzyme immunoassay is extremely versatile in permitting spectrophotometric determinations. The immunoassay employs an enzyme to which the organic compound to be determined is conjugated. The organic compound is conjugated at a position where, when bound to antibody, the activity of the enzyme is substantially reduced. To the extent that the unknown sample contains the same organic compound, the amount of antibody available for binding to the organic compound conjugated to the enzyme is reduced. Therefore, by analyzing for enzymatic activity, a significant increase in enzymatic activity over the enzymatic activity in the absence of the unknown indicates the presence of the organic compound in the unknown.
The sensitivity of the homogeneous enzyme immunoassay is based to a substantial degree on the activity of the enzyme when conjugated and the degree of inhibitability when antibody is bound to the organic compound conjugated to the enzyme. It is, therefore, desirable to have an enzyme which not only has a high turnover rate initially, but retains a substantial proportion of this high turnover rate after conjugation, and is strongly inhibited when antibody is bound to the organic compound which is conjugated to the enzyme. Also, the enzyme should allow for strong specific binding of antibody to the conjugated organic compound.